This proposal seeks to elucidate the pathophysiology and pathogenesis of two clinically important states of contractile dysfunction: stunned myocardium and failing myocardium. In the original funding period, magnetic resonance spectroscopy was used to characterize excitation- contraction (E-C) coupling in hypoxic, ischemic, hibernating and stunned hearts. We found that reversible post-ischemic dysfunction (stunning) is due not to a deficiency of activator Ca2+, but rather to reduced Ca2+ responsiveness of the contractile proteins. Various lines of evidence, including direct measurements of [Ca2+]i during ischemia and reperfusion, led us to hypothesize that stunning reflects damage to the contractile proteins by Ca2+ -activated proteases which are turned on in the initial moments of reflow. This hypothesis attractively explains many features of stunning including its distinctive time course of recovery (days to weeks), which is consistent with the time that would be required to synthesize new contractile proteins for the repair of damaged myofilaments. The use of NMR enabled unique estimates of [Ca2+]i in perfused hearts, but with limited experimental flexibility. We now propose to extend our characterization of contractile dysfunction from the organ level to the cellular and molecular levels. The method of Backx and ter Keurs will be used to measure contractile force and intracellular free ion concentrations in small multicellular preparations (trabeculae) from rat ventricle. Ca2+ transients and steady-state [Ca2+]i-force relationships will be determined in intact muscles; crossbridge kinetics will be characterized in further detail in the same trabeculae after chemical skinning. The methods will be adapted to enable the characterization of E-C coupling not only in control muscles but also in those from stunned and failing hearts (the SHHF/Mcc-cp rat heart failure model). To test the proteolytic hypothesis of stunning, we will determine the structural and functional consequences of exposure of the myofilaments to Ca2+-activated proteases. Biochemical and molecular genetic methods will be used to determine whether or not calpains are activated in the stunned myocardium, to verify that proteolysis occurs and to determine which specific contractile proteins are degraded. The findings with stunning, in which the myofilaments have been implicated, will be compared and contrasted to those in failing myocardium, in which altered Ca2+ handling by the sarcoplasmic reticulum appears to play a dominant role. These lines of investigation promise to advance our basic understanding of cardiac contractile function in health and disease.